Leukotriene B4 (LTB4)
Leukotriene B4 is produced by leukocytes, particularly macrophage and monocytes upon activation by immune complexes, phagocytosis or other stimuli. In this process, membrane phospholipids are broken down by phospholipase A2 to release arachidonic acid, which is further metabolized via one of two pathways. The first is via cycloxygenases to produce prostaglandins. The second is via lipoxygenases to form leukotriene A4 (LTA4). LTA4 is converted to LTB4 or LTC4. LTB4 is a potent chemotactic agent that stimulates neutrophil and macrophage migration (chemotaxis) to sites of inflammation. The structure of LTB4 is shown below. 
The known pathophysiological responses of LTB4 include: induction of potent neutrophil chemotactic activity, promotion of adhesion of polymorphonuclear leukocytes (PMN's) to vasculature, increase in vascular permeability, stimulation of the release of lysosomal enzymes, by PMN's. The pro-inflammatory action of LTB4 has been demonstrated in vivo, wherein topical LTB4 on human skin promotes the infiltration of PMN's and other inflammatory cells. Intradermal injection of LTB4 induces accumulation of neutrophils at the injection site. Intravenous injection of LTB4 causes rapid but transient neutropenia (Kingsbury et al., J. Med. Chem., 1993, 36, 3308-3320; and references cited therein).
In addition, the presence of physiologically relevant LTB4 concentration at inflammatory sites has been associated with, for example, disease states such as psoriasis, asthma and active gout; in colonic mucosa associated with inflammatory bowel disease; in synovial fluid from patients with active rheumatoid arthritis; and in reperfusion injury. All of these observations together support the involvement of LTB4 in human inflammatory disease (Kingsbury et al, and Griffeths et al., Proc. Natl. Acad. Sci. Vol. 92, pp517-521, January 1995; and references cited therein.).
LTB4 is believed to interact with two sub-groups of receptor: a high-affinity receptor and a low-affinity receptor. Research indicates that the high-affinity receptor mediates chemotaxis and that the low-affinity receptor mediates LTB4-induced secretory and oxidase-activation responses (Yokomizo et al. 2000). Some LTB4 antagonists are observed to antagonize all LTB4 mediated activity. Other LTB4 antagonists modulate only the activity associated with one but not the other sub-population of LTB4 receptors.
LTB4 Antagonists
Compounds, which act as antagonists of LTB4 include, for example: structural analogs of LTB4 such as LTB4-dimethyl amide and 20-CF3-LTB4; SM-9064; U-75302; Ly-223982; SC-41930; ONO 4057 (Prostaglandins, 44(4):261-275, 1992); RG-14893; (E)-3-[2-[6-[3-(3-butoxyphenyl)-3-hydroxypropenyl]pyridin-2-yl]-1-hydroxyethyl]benzoate-benzoic acid, lithium salt (Kingsbury J. Med. Chem, 1993, 36, 3308-3320, and references cited therein); the natural product Leucettamine A and a structural analog, 1-methyl-2-amino-4-[[4′-[4″-(hydroxybutyl)phenyl]methyl]-5-(phenyl-methyl)imidazole (Boehm et al, J. Med. Chem, 1993, 36, 22, 3333-3340); a series of pyridine-2-acrylic acids (Kingsbury et al., J. Med. Chem., 1993, 36, 22, 3321-3332); SC-45694 (Tsai et al, J. Pharm.Exp.Ther., 268, 3, 1493-1498); a series of essential fatty acids (Yagaloff et al., Prostaglandins, Leukotrienes and Essential Fatty Acids (1995), 52, 293-297); and FPL 55712 and FPL 55231 (Cheng et al., J. Pharm. Exp. Ther., 236(1), 1985). The structures of these compounds show many similarities to the structure of LTB4.
2,3-Benzodiazepines
Certain 2,3-benzodiazepines have been explored extensively for their potent CNS modulating activity. Compounds such as tofisopam (Grandaxin®), girisopam, and norisopam have demonstrated substantial anxiolytic and antipsychotic activity.
Tofisopam has been shown in humans to have an activity profile that is significantly different from that of widely used 1,4-benzodiazepine (BZ) anxiolytics such as diazepam (Valium®) and chlordiazepepoxide (Librium®). The 1,4-benzodiazepine, in addition to having sedative-hypnotic activity, also possess muscle relaxant and anticonvulsant properties which, though therapeutically useful in some disease states, are nonetheless potentially untoward side effects. Thus, the 1,4-benzodiazepines, though safe when administered alone, may be dangerous in combination with other CNS drugs including alcohol.
Tofisopam, in contrast, is a non-sedative anxiolytic that has no appreciable sedative, muscle relaxant or anticonvulsant properties (Horvath et al., Progress in Neurobiology, 60 (2000), 309-342). In clinical studies, tofisopam improved rather than impaired psychomotor performance and showed no interaction with ethanol (Id.). These observations comport with data that show that tofisopam does not interact with central BZ receptors and binds only weakly to peripheral BZ receptors. Additional studies have shown that tofisopam enhances mitogen-induced lymphocyte proliferation and IL-2 production in vitro.
Other 2,3-benzodiazepines that are structurally similar to tofisopam have been investigated and shown to have varying activity profiles. For example, GYKI-52466 and GYKI-53655 (structures shown below) act as noncompetitive glutamate antagonists at the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) site, and have demonstrated neuroprotective, muscle relaxant and anticonvulsant activity (Id.). Another group of 2,3-benzodiazepines that have been investigated are represented by the compound GYKI-52895, and show activity as selective dopamine uptake inhibitors with potential use in antidepressant and anti-Parkinsonism therapy.
Tofisopam (structure shown below), with the atom numbering system indicated) is a racemic mixture of (R)- and (S)-enantiomers. This is due to the asymmetric carbon, i.e., a carbon with four different groups attached, at the 5-position of the benzodiazepine ring. 
The molecular structure and conformational properties of tofisopam have been determined by NMR, CD and x-ray crystallography (Visy et al., Chirality 1:271-275 (1989)). The 2,3-diazepine ring exists as two different conformers. The major tofisopam conformers, (+)R and (−)S, contain a 5-ethyl group in a quasi-equatorial position. The 5-ehtyl group is positioned quasi-axially in the minor conformers, (−)R and (+)S. Thus, racemic tofisopam can exist as four molecular species, i.e., two enantiomers, each of which exists as two conformations. The sign of the optical rotation is reversed upon inversion of the diazepine ring from one conformer to the other. In crystal form, tofisopam exists only as the major conformations, with dextrorotatory tofisopam being of the (R) absolute configuration. (Toth et al., J. Heterocyclic Chem., 20:709-713 (1983); Fogassy et al., Bioorganic Heterocycles, Van der Plas, H. C., Ötvös, L, Simongi, M., eds. Budapest Amsterdam: Akademia; Kiado-Elsevier, 229:233 (1984)).
Differential binding of the (+) and (−) conformations of tofisopam has been reported in binding studies with human albumin (Simongi et al. Biochem. Pharm., 32(12), 1917-1920, 1983). The two (+/−) conformers have also been reported as existing in equilibrium (Zsila et al., Journal of Liquid Chromatography & Related Technologies, 22(5), 713-719, 1999; and references therein).
The optically pure (R)-enantiomer of tofisopam (R)-1-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7,8-dimethoxy-5H-2,3-benzodiazepine) has been isolated and shown to possess the nonsedative anxiolytic activity of the racemic mixture. See U.S. Pat. No. 6,080,736; the entire disclosure of which is incorporated herein by reference.
Inflammatory Disorders
Crohn's disease and ulcerative colitis, collectively referred to as inflammatory bowel disease (IBD), are chronic recurrent inflammatory diseases of unclear etiology, affecting the small intestine and colon. Inflammatory bowel disease (IBD) can involve either or both the small and large bowel. These disorders fall into the category of “idiopathic” inflammatory bowel disease because the etiology for them is unknown.
Pathologic findings are generally not specific, although they may suggest a particular form of IBD. “Active” IBD is characterized by acute inflammation. “Chronic” IBD is characterized by architectural changes of crypt distortion and scarring. The term “crypt” refers to a deep pit that protrudes down into the connective tissue surrounding the small intestine. Crypt abscesses (active IBD characterized by the presence of neutrophils in crypt lumens) can occur in many forms of IBD, not just ulcerative colitis. Under normal conditions the epithelium at the base of the crypt is the site of stem cell proliferation and the differentiated cells move upwards and are shed 3-5 days later at the tips of the villi. This normal process, necessary for proper bowel function, is interrupted by IBD
Ulcerative colitis (UC) involves the colon as a diffuse mucosal disease with distal predominance. The rectum is virtually always involved, and additional portions of colon may be involved extending proximally from the rectum in a continuous pattern. Most often ulcerative colitis occurs in young people 15 to 40 years of age. Ulcerative colitis occurs only in the inner lining of the colon (large intestine) or rectum. When it is localized in the rectum, it is called “proctitis”.
Crohn's Disease is a chronic inflammatory disease that has periods of remission (time when person feels well) and relapse (when a person feels ill). Crohn's disease is an inflammation and ulceration process that occurs in the deep layers of the intestinal wall. The most common areas affected are the lower part of the small intestine, called the ileum, and the first part of the colon. This type of Crohn's disease is called ileocolitis. Crohn's disease can infrequently affect any part of the upper gastrointestinal tract. Aphthous ulcers, which are similar to cold sores, are common. Ulcers can also occur in the esophagus, stomach and duodenum.
Therapy for IBD has historically included administration of corticosteroids. However drawbacks of long term corticosteroid therapy include masking (or induction) of intestinal perforation, osteonecrosis and metabolic bone disease. Additional problems relate to development of corticosteroid dependency (Habnauer, New England Journal of Medicine, 334(13), p 841-848, 1996). Aminosalicylates such as sulfasalazine and mesalamine have been used to treat mild or moderately active ulcerative colitis and Crohn's Disease, and to maintain remission (Id at 843). Immunomodulatory drugs such as azathioprine and mercaptopurine have been used in long term treatment for patients with IBD. Common complications with both of these drugs include pancreatitis, which occurs with an incidence of 3-15% of patients, and bone marrow suppression, which requires regular monitoring. More potent immunosuppressive drugs such as cyclosporine and methotrexate have been employed, but toxicity of these drugs limits their use to specific situations of refractory disease states. Other therapeutic approaches include antibiotic therapy and nutritional therapy. Often, therapy involves a combination of the above-described drug therapies in addition to surgical resection of the bowel.
There is no cure for IBD. Ultimately, the chronic and progressive nature of IBD demands a long-term treatment that maximizes the local antiinflammatory effect while minimizing the global systemic effect on the immune system.
Chronic inflammatory disorders such as Crohn's Disease typically demonstrate periods of remission between intervals when the inflammatory is active and requires acute treatment. This is an example of a circumstance wherein it is known beforehand that an individual will develop, or is likely to develop an inflammatory disorder.
Another chronic inflammatory condition believed to be mediated by LTB4 is psoriasis. Psoriasis is a chronic, recurrent, papulosquamous plaque on areas of trauma such as the elbow, knee or scalp, though it may appear elsewhere on the skin. Psoriasis may coexist with lupus erythematosis in some individuals. Current treatments include topical administration of psoralens. “Psoralens” refers to a group of substances found in many different plants, especially psoralea corylifolia. Psoralens interact with nucleic acids and are also used as research tools. Psoriasis is also treated by long-wave ultraviolet radiation. Neither treatment cures or prevents recurrence of psoriasis symptoms.
Another chronic inflammatory disorder believed to be mediated by LTB4 is rheumatoid arthritis, which is an autoimmune disease of the joints. Rheumatoid arthritis is characterized by the following criteria 1-7, wherein criteria 1-4 are present for more than 6 weeks: (1) morning stiffness in and around joints lasting at least one hour before maximum improvement; (2) soft tissue swelling (arthritis) of three or more joints observed by a physician; (3) swelling (arthritis) of the proximal interphalangeal, metacarpal phalangeal, or wrist joints; (4) symmetric swelling; (5) rheumatoid nodules, i.e., a granulomatous lesion characterized by central necrosis encircled by a palisade of monocytes and an exterior mantle of lymphocytic infiltrate. These lesions present as subcutaneous nodules, especially at pressure points such as the elbow in individuals with rheumatoid arthritis or other rheumatoid disorders; (6) presence of rheumatoid factors, i.e., an autoantibody in the serum of individuals with rheumatoid arthritis; and (7) roentgenographic erosions, i.e., joint lesions visible on an X-ray.
Rheumatoid arthritis is a chronic disorder for which there is no known cure. The major goals of treatment of rheumatoid arthritis are to reduce pain and discomfort, prevent deformities and loss of joint function, and maintain a productive and active life. Inflammation must be suppressed and mechanical and structural abnormalities corrected or compensated by assistive devices. Treatment options include reduction of joint stress, physical and occupational therapy, drug therapy, and surgical intervention.
There are three general classes of drugs commonly used in the treatment of rheumatoid arthritis: non-steroidal anti-inflammatory agents (NSAID's), corticosteroids, and remittive agents or disease modifying anti-rheumatic drugs (DMARD's). NSAID's and corticosteroids have a short onset of action while DMARD's can take several weeks or months to demonstrate a clinical effect. DMARD's include leflunomide (Arava™), etanercept (Enbrel™), infliximab (Remicade™), antimalarials, methotrexate, gold salts, sulfasalazine, d-penicillamine, cyclosporin A, cyclophosphamide and azathioprine. Because cartilage damage and bony erosions frequently occur within the first two years, rheumatologists now move more aggressively to a DMARD agent.
Treatmant of rheumatoid arthritis by chronic administration of a corticosteroid involves the same side effect profile as discussed regarding IBD above. Chronic administration of NSAID's also produces side effects. The most common toxicity of NSAID's is gastrointestinal disturbance. Because prostaglandins play a role in the regulation of renal blood flow and maintenance of glomerular filtration, NSAID's can impair renal function in certain patients. Weight gain and cushingoid appearance is a frequent problem and source of patient complaints. Recent studies have raised concern over the increased cardiovascular risk and accelerated osteoporosis associated with low dose prednisone particularly at doses above 10 mg daily.
Gout is another inflammatory disorder believed to be mediated by LTB4. Gout is characterized by a disturbance of uric-acid metabolism occurring chiefly in males. Gout is characterized by painful inflammation of the joints, especially of the feet and hands, and arthritic attacks resulting from elevated levels of uric acid in the blood and the deposition of urate crystals around the joints. The condition can become chronic and result in deformity.
Gout can present another circumstance wherein it is known beforehand that an individual will or is likely to develop an inflammatory disorder. In the instance of patients undergoing radiotherapy or chemotherapy, the individual may experience a dramatic rise in serum uric acid levels associated with lysis of the tumor mass. Such large increases in uric acid can deposit urate crystals in synovial fluid of joints thereby causing the inflammatory disorder, gout. When such a rise in serum uric acid levels is known to be likely, prophylaxis with an LTB4 antagonist can act to prevent the inflammatory condition of gout.
Radiation-induced gastrointestinal inflammation is another inflammatory disorder believed to be mediated by LTB4. Radiation works by damaging cancer cells, but unfortunately can damage non-diseased tissue as well, causing a typical inflammatory reaction in response. Therapeutic radiation is thus generally applied to a defined area of the subject's body which contains abnormal proliferative tissue in order to maximize the dose absorbed by the abnormal tissue and minimize the dose absorbed by the nearby normal tissue. However, it is difficult (if not impossible) to selectively administer therapeutic ionizing radiation to the abnormal tissue. Thus, normal tissue proximate to the abnormal tissue is also exposed to potentially damaging doses of ionizing radiation throughout the course of treatment. Moreover, some treatments that require exposure of the subject's entire body to the radiation, in a procedure called “total body irradiation”, or “TBI.” The efficacy of radiotherapeutic techniques in destroying abnormal proliferative cells is therefore necessarily balanced by the associated cytotoxic effects on nearby normal cells.
After or during a course of radiotherapy, LTB4-mediated inflammatory processes may be triggered, causing damage to the bowel, and leading to sloughing of the cells of the inner lining of the GI tract. Radiation-induced gastrointestinal inflammation can present another circumstance wherein it is known beforehand that an individual will or is likely to develop an inflammatory disorder. In the instance of patients undergoing radiotherapy, the inflammation, damage and sloughing of the gastrointestinal tract is a predictable side effect of the radiotherapy.
New antiinflammatory agents are needed which are useful in the treatment of inflammatory disorders such as IBD, rheumatoid arthritis, gout, psoriasis and radiation-induced gastrointestinal inflammation. In particular, agents are needed that are appropriate for chronic long-term use in treatment. In addition, agents are needed that are useful in the prevention of LTB4-mediated inflammatory disorders that occur secondary to observable events such as ionizing radiation therapy.